Cas13d-mediated multiplex RNA targeting confers a broad-spectrum resistance against RNA viruses in potato.

CRISPR-Cas systems endow the bacterial and archaeal species with adaptive immune mechanisms to fend off invading phages and foreign plasmids. The class 2 type VI CRISPR/Cas effector Cas13d has been harnessed to confer the protection against RNA viruses in diverse eukaryotic species. However a vast number of different viruses can potentially infect the same host plant resulting in mixed infection, thus necessitating the generation of crops with broad-spectrum resistance to multiple viruses. Here we report the repurposing of CRISPR/Cas13d coupled with an endogenous tRNA-processing system (polycistronic tRNA-gRNA, PTG) to target the multiple potato RNA viruses. Expression of Cas13d and four different gRNAs were observed in transgenic potato lines expressing the Cas13d/PTG construct. We show that the Cas13d/PTG transgenic plants exhibit resistance to either PVY, PVS, PVX or PLRV alone or two/three viruses simultaneously by reducing viral accumulation in plant cells. In sum, our findings provide an efficient strategy for engineering crops that can simultaneously resist infection by multiple RNA viruses.

Dietary flavonoid narirutin as a prospective antagonist of oncogenic pri/pre-microRNAs.

MicroRNAs (miRNAs) are involved in cancer progression via translational degradation in a sequence-specific manner of the 3'-untranslated region (3'UTR) of messenger RNA (mRNA). The involvement of miRNA in the biological progression of various cancer types is considered to be a potential target. Primary miRNA (pri-miRNA) and precursor-miRNA (pre-miRNA) synthesize the miRNA by dicer-catalyzed processes thus targeting pri/pre-miRNA by phytochemicals is amongst the appropriate approaches for anticancer therapies. Flavonoids category of phytochemicals is well-known for its chemotherapeutic and chemopreventive potential against multiple cancer types. However, the molecular interactions of flavonoids with miRNAs are not reported so far. Thus, this study aims to identify the promising flavonoids as the antagonist of miRNAs (pre-miR21, pri-miR-208a, pri-miR-378a, pri-miR320b, pri-miR-300, pri-miR-19b, and pre-miR-20b) using molecular docking simulations studies. Among the tested flavonoids, narirutin showed highest binding energy (-11.7 kcal/mol) against pri-miR19b followed by pri-miR-378a (-11.4 kcal/mol) > pri-miR320b (-11.2 kcal/mol) = pri-miR-300 (-11.2 kcal/mol) > pri-miR-208a (-9.0 kcal/mol) > pre-miR-20b (- 8.3 kcal/mol). The molecular dynamic simulation experiment confirmed that narirutin destabilizes the tertiary structure of pri-miRNA in comparison to apo-RNA. The finding indicates that narirutin binding with pre-miRNA causes disruption of pri-RNA structure that creates a loss of DICER-pre-miRNA interactions by hindering the pre-miRNA synthesis, thereby affecting miRNA processing. Further pharmacokinetics and toxicity prediction revealed that it is non-carcinogenic, non-mutagenic, and does not inhibit the CYPs activity. Thus, narirutin could be a possible antagonist of oncogenic miRNAs, therefore could be useful for miRNA-targeted cancer prevention and treatment.

Long non-coding RNA Xist regulates oocyte loss via suppressing miR-23b-3p/miR-29a-3p maturation and upregulating STX17 in perinatal mouse ovaries.

The fecundity of female mammals is resolved by the limited size of the primordial follicle (PF) pool formed perinatally. The establishment of PF pool is accompanied by a significant programmed oocyte death. Long non-coding RNAs (lncRNA) are central modulators in regulating cell apoptosis or autophagy in multiple diseases, however, the significance of lncRNAs governing perinatal oocyte loss remains unknown. Here we find that Yin-Yang 1 (YY1) directly binds to the lncRNA X-inactive-specific transcript (Xist) promoter and facilitates Xist expression in the perinatal mouse ovaries. Xist is highly expressed in fetal ovaries and sharply downregulated along with the establishment of PF pool after birth. Gain or loss of function analysis reveals that Xist accelerates oocyte autophagy, mainly through binding to pre-miR-23b or pre-miR-29a in the nucleus and preventing the export of pre-miR-23b/pre-miR-29a to the cytoplasm, thus resulting in decreased mature of miR-23b-3p/miR-29a-3p expression and upregulation miR-23b-3p/miR-29a-3p co-target, STX17, which is essential for timely control of the degree of oocyte death in prenatal mouse ovaries. Overall, these findings identify Xist as a key non-protein factor that can control the biogenesis of miR-23b-3p/miR-29a-3p, and this YY1-Xist-miR-23b-3p/miR-29a-3p-STX17 regulatory axis is responsible for perinatal oocyte loss through autophagy.

Snord116 Post-transcriptionally Increases Nhlh2 mRNA Stability: Implications for Human Prader-Willi Syndrome.

The smallest genomic region causing Prader-Willi Syndrome (PWS) deletes the non-coding RNA SNORD116 cluster; however, the function of SNORD116 remains a mystery. Previous work in the field revealed the tantalizing possibility that expression of NHLH2, a gene previously implicated in both obesity and hypogonadism, was downregulated in PWS patients and differentiated stem cells. In silico RNA: RNA modeling identified several potential interaction domains between SNORD116 and NHLH2 mRNA. One of these interaction domains was highly conserved in most vertebrate NHLH2 mRNAs examined. A construct containing the Nhlh2 mRNA, including its 3'-UTR, linked to a c-myc tag was transfected into a hypothalamic neuron cell line in the presence and absence of exogenously-expressed Snord116. Nhlh2 mRNA expression was upregulated in the presence of Snord116 dependent on the length and type of 3'UTR used on the construct. Furthermore, use of actinomycin D to stop new transcription in N29/2 cells demonstrated that the upregulation occurred through increased stability of the Nhlh2 mRNA in the 45 minutes immediately following transcription. In silico modeling also revealed that a single nucleotide variant (SNV) in the NHLH2 mRNA could reduce the predicted interaction strength of the NHLH2:SNORD116 diad. Indeed, use of an Nhlh2 mRNA construct containing this SNV significantly reduces the ability of Snord116 to increase Nhlh2 mRNA levels. For the first time, these data identify a motif and mechanism for SNORD116-mediated regulation of NHLH2, clarifying the mechanism by which deletion of the SNORD116 snoRNAs locus leads to PWS phenotypes.

A single nucleotide substitution in the coding region of Ogura male sterile gene, orf138, determines effectiveness of a fertility restorer gene, Rfo, in radish.

Cytoplasmic male sterility (CMS) observed in many plants leads defect in the production of functional pollen, while the expression of CMS is suppressed by a fertility restorer gene in the nuclear genome. Ogura CMS of radish is induced by a mitochondrial orf138, and a fertility restorer gene, Rfo, encodes a P-type PPR protein, ORF687, acting at the translational level. But, the exact function of ORF687 is still unclear. We found a Japanese variety showing male sterility even in the presence of Rfo. We examined the pollen fertility, Rfo expression, and orf138 mRNA in progenies of this variety. The progeny with Type H orf138 and Rfo showed male sterility when their orf138 mRNA was unprocessed within the coding region. By contrast, all progeny with Type A orf138 were fertile though orf138 mRNA remained unprocessed in the coding region, demonstrating that ORF687 functions on Type A but not on Type H. In silico analysis suggested a specific binding site of ORF687 in the coding region, not the 5' untranslated region estimated previously, of Type A. A single nucleotide substitution in the putative binding site diminishes affinity of ORF687 in Type H and is most likely the cause of the ineffectiveness of ORF687. Furthermore, fertility restoration by RNA processing at a novel site in some progeny plants indicated a new and the third fertility restorer gene, Rfs, for orf138. This study clarified that direct ORF687 binding to the coding region of orf138 is essential for fertility restoration by Rfo.

LC-MS/MS Profiling of Post-Transcriptional Modifications in Ginseng tRNA Purified by a Polysaccharase-Aided Extraction Method.

Transfer RNAs (tRNAs) are the most heavily modified RNA species in life entities. Post-transcriptional modifications severely impact the structure and function of tRNAs. To date, hundreds of modifications have been identified in tRNAs, mainly from microorganisms and animals. However, tRNAs in plant roots or tubers that have been widely used for food and medical purpose for centuries are rarely studied because isolation of RNA from plants still remains a challenge. In this paper, a polysaccharase-aided RNA isolation (PARI) method for extraction of high-quality RNA from plants containing large quantities of polysaccharides is developed. This method presents a new strategy of "digesting" polysaccharides that is completely different from the conventional method of "dissolving" the contaminants. By using this method, RNA of high integrity and purity were successfully extracted from ginseng roots because polysaccharide contaminations were removed efficiently with α-amylase digestion. Ginseng tRNAs were first sequenced by NGS and a total of 41 iso acceptors were identified. ChloroplastictRNAGly(GCC) in ginseng root was purified and four modified nucleosides, including m7G, D, T, and Ψ, were identified by LC-MS/MS. The results also revealed that the m7G occurs at a novel position 18, which may be related to the deformation of D-loop. PARI is the first enzyme-assisted technique for RNA isolation from plants, which could fundamentally solve the problem of polysaccharide contaminations. By using the PARI method, more individual tRNAs could be isolated easily from polysaccharide-rich plant tissues, which would have a positive impact on the feasibility of research on structure and function of tRNA in plants.

Post-transcriptional regulation activity through alternative splicing involved in the effects of Aloe vera on the Wnt/ß-catenin and Notch pathways in colorectal cancer cells.

Aloe vera (L.) Burm.f. is widely used as laxative drugs, cosmetics, and functional food due to a variety of therapeutic effects. However, several studies indicated a colonic carcinogenic activity of Aloe vera. But the underline mechanism has not been well clarified. This study aimed to explore the potential mechanism at the post-transcriptional level. Identification of Differential Expressed Alternative Splicing (DEAS) genes and events and the corresponding functional enrichment analyses were conducted on RKO cells after treated with Aloe vera aqueous extract and its two active components, aloin and aloesin. And RT-qPCR was conducted for validation. Results indicated that they induced 2200, 2342 and 2133 DEAS events, respectively. The GO enrichment and the COG classification results of DEAS genes showed that they were associated with transcription, as well as functions like signal transduction mechanisms. Moreover, DEAS genes related to the two colorectal cancerous pathways, Wnt and Notch pathways, were annotated. In conclusion, aloe extract, aloin and aloesin significantly regulated the DEAS profile of RKO cells. The colonic carcinogenicity of Aloe vera may due to its post-transcriptional regulatory activity through Alternative Splicing (AS) on genes, especially on Wnt-related and Notch-related key genes.

Epitranscriptomic systems regulate the translation of reactive oxygen species detoxifying and disease linked selenoproteins.

Here we highlight the role of epitranscriptomic systems in post-transcriptional regulation, with a specific focus on RNA modifying writers required for the incorporation of the 21st amino acid selenocysteine during translation, and the pathologies linked to epitranscriptomic and selenoprotein defects. Epitranscriptomic marks in the form of enzyme-catalyzed modifications to RNA have been shown to be important signals regulating translation, with defects linked to altered development, intellectual impairment, and cancer. Modifications to rRNA, mRNA and tRNA can affect their structure and function, while the levels of these dynamic tRNA-specific epitranscriptomic marks are stress-regulated to control translation. The tRNA for selenocysteine contains five distinct epitranscriptomic marks and the ALKBH8 writer for the wobble uridine (U) has been shown to be vital for the translation of the glutathione peroxidase (GPX) and thioredoxin reductase (TRXR) family of selenoproteins. The reactive oxygen species (ROS) detoxifying selenocysteine containing proteins are a prime examples of how specialized translation can be regulated by specific tRNA modifications working in conjunction with distinct codon usage patterns, RNA binding proteins and specific 3' untranslated region (UTR) signals. We highlight the important role of selenoproteins in detoxifying ROS and provide details on how epitranscriptomic marks and selenoproteins can play key roles in and maintaining mitochondrial function and preventing disease.

A screening platform to monitor RNA processing and protein-RNA interactions in ribonuclease P uncovers a small molecule inhibitor.

Ribonucleoprotein (RNP) complexes and RNA-processing enzymes are attractive targets for antibiotic development owing to their central roles in microbial physiology. For many of these complexes, comprehensive strategies to identify inhibitors are either lacking or suffer from substantial technical limitations. Here, we describe an activity-binding-structure platform for bacterial ribonuclease P (RNase P), an essential RNP ribozyme involved in 5' tRNA processing. A novel, real-time fluorescence-based assay was used to monitor RNase P activity and rapidly identify inhibitors using a mini-helix and a pre-tRNA-like bipartite substrate. Using the mini-helix substrate, we screened a library comprising 2560 compounds. Initial hits were then validated using pre-tRNA and the pre-tRNA-like substrate, which ultimately verified four compounds as inhibitors. Biolayer interferometry-based binding assays and molecular dynamics simulations were then used to characterize the interactions between each validated inhibitor and the P protein, P RNA and pre-tRNA. X-ray crystallographic studies subsequently elucidated the structure of the P protein bound to the most promising hit, purpurin, and revealed how this inhibitor adversely affects tRNA 5' leader binding. This integrated platform affords improved structure-function studies of RNA processing enzymes and facilitates the discovery of novel regulators or inhibitors.

Alternative splicing coupled to nonsense-mediated mRNA decay contributes to the high-altitude adaptation of maca (Lepidium meyenii).

Alpine plants remain the least studied plant communities in terrestrial ecosystems. However, how they adapt to high-altitude environments is far from clear. Here, we used RNA-seq to investigate a typical alpine plant maca (Lepidium meyenii) to understand its high-altitude adaptation at transcriptional and post-transcriptional level. At transcriptional level, we found that maca root significantly up-regulated plant immunity genes in day-time comparing to night-time, and up-regulated abiotic (cold/osmotic) stress response genes in Nov and Dec comparing to Oct. In addition, 17 positively selected genes were identified, which could be involved in mitochondrion. At post-transcriptional level, we found that maca had species-specific characterized alternative splicing (AS) profile which could be influenced by stress environments. For example, the alternative 3' splice site events (A3SS, 39.62%) were predominate AS events in maca, rather than intron retention (IR, 23.17%). Interestingly, besides serine/arginine-rich (SR) proteins and long non-coding RNAs (lncRNAs), a lot of components in nonsense-mediated mRNA decay (NMD) were identified under differential alternative splicing (DAS), supporting AS coupled to NMD as essential mechanisms for maca's stress responses and high-altitude adaptation. Taken together, we first attempted to unveil maca's high-altitude adaptation mechanisms based on transcriptome and post-transcriptome evidence. Our data provided valuable insights to understand the high-altitude adaptation of alpine plants.

Neuropeptidome of the Hypothalamus and Pituitary Gland of Indicine × Taurine Heifers: Evidence of Differential Neuropeptide Processing in the Pituitary Gland before and after Puberty.

Puberty in cattle is regulated by an endocrine axis, which includes a complex milieu of neuropeptides in the hypothalamus and pituitary gland. The neuropeptidome of hypothalamic-pituitary gland tissue of pre- (PRE) and postpubertal (POST) Bos indicus-influenced heifers was characterized, followed by quantitative analysis of 51 fertility-related neuropeptides in these tissues. Comparison of peptide abundances with gene expression levels allowed assessment of post-transcriptional peptide processing. On the basis of classical cleavage, 124 mature neuropeptides from 35 precursor proteins were detected in hypothalamus and pituitary gland tissues of three PRE and three POST Brangus heifers. An additional 19 peptides (cerebellins, PEN peptides) previously reported as neuropeptides that did not follow classical cleavage were also identified. In the pre-pubertal hypothalamus, a greater diversity of neuropeptides (25.8%) was identified relative to post-pubertal heifers, while in the pituitary gland, 38.6% more neuropeptides were detected in the post-pubertal heifers. Neuro-tissues of PRE and POST heifers revealed abundance differences ( p < 0.05) in peptides from protein precursors involved in packaging and processing (e.g., the granin family and ProSAAS) or neuron stimulation (PENK, CART, POMC, cerebellins). On their own, the transcriptome data of the precursor genes could not predict the neuropeptide profile in the exact same tissues in several cases. This provides further evidence of the importance of differential processing of the neuropeptide precursors in the pituitary before and after puberty.

Elongator subunit 3 (ELP3) modifies ALS through tRNA modification.

Amyotrophic lateral sclerosis (ALS) is a fatal degenerative motor neuron disorder of which the progression is influenced by several disease-modifying factors. Here, we investigated ELP3, a subunit of the elongator complex that modifies tRNA wobble uridines, as one of such ALS disease modifiers. ELP3 attenuated the axonopathy of a mutant SOD1, as well as of a mutant C9orf72 ALS zebrafish model. Furthermore, the expression of ELP3 in the SOD1G93A mouse extended the survival and attenuated the denervation in this model. Depletion of ELP3 in vitro reduced the modified tRNA wobble uridine mcm5s2U and increased abundance of insoluble mutant SOD1, which was reverted by exogenous ELP3 expression. Interestingly, the expression of ELP3 in the motor cortex of ALS patients was reduced and correlated with mcm5s2U levels. Our results demonstrate that ELP3 is a modifier of ALS and suggest a link between tRNA modification and neurodegeneration.

Modulation of oncogenic miRNA biogenesis using functionalized polyamines.

MicroRNAs are key factors in the regulation of gene expression and their deregulation has been directly linked to various pathologies such as cancer. The use of small molecules to tackle the overexpression of oncogenic miRNAs has proved its efficacy and holds the promise for therapeutic applications. Here we describe the screening of a 640-compound library and the identification of polyamine derivatives interfering with in vitro Dicer-mediated processing of the oncogenic miR-372 precursor (pre-miR-372). The most active inhibitor is a spermine-amidine conjugate that binds to the pre-miR-372 with a KD of 0.15 µM, and inhibits its in vitro processing with a IC50 of 1.06 µM. The inhibition of miR-372 biogenesis was confirmed in gastric cancer cells overexpressing miR-372 and a specific inhibition of proliferation through de-repression of the tumor suppressor LATS2 protein, a miR-372 target, was observed. This compound modifies the expression of a small set of miRNAs and its selective biological activity has been confirmed in patient-derived ex vivo cultures of gastric carcinoma. Polyamine derivatives are promising starting materials for future studies about the inhibition of oncogenic miRNAs and, to the best of our knowledge, this is the first report about the application of functionalized polyamines as miRNAs interfering agents.

Eurycomalactone Inhibits Expression of Endothelial Adhesion Molecules at a Post-Transcriptional Level.

The C-19 quassinoid eurycomalactone (1) has recently been shown to be a potent (IC50 = 0.5 µM) NF-κB inhibitor in a luciferase reporter model. In this study, we show that 1 with similar potency inhibited the expression of the NF-κB-dependent target genes ICAM-1, VCAM-1, and E-selectin in TNFα-activated human endothelial cells (HUVECtert) by flow cytometry experiments. Surprisingly, 1 (2 µM) did not inhibit TNFα-induced IKKα/ß or IκBα phosphorylation significantly. Also, the TNFα-induced degradation of IκBα remained unchanged in response to 1 (2 µM). In addition, pretreatment of HUVECtert with 1 (2 µM) had no statistically significant effect on TNFα-mediated nuclear translocation of the NF-κB subunit p65 (RelA). Quantitative RT-PCR revealed that 1 (0.5-5 µM) exhibited diverse effects on the TNFα-induced transcription of ICAM-1, VCAM-1, and SELE genes since the mRNA level either remained unchanged (ICAM-1, E-selectin, and VCAM-1 at 0.5 µM 1), was reduced (VCAM-1 at 5 µM 1), or even increased (E-selectin at 5 µM 1). Finally, the time-dependent depletion of a short-lived protein (cyclin D1) as well as the measurement of de novo protein synthesis in the presence of 1 (2-5 µM) suggested that 1 might act as a protein synthesis inhibitor rather than an inhibitor of early NF-κB signaling.

Solanum tuberosum StCDPK1 is regulated by miR390 at the posttranscriptional level and phosphorylates the auxin efflux carrier StPIN4 in vitro, a potential downstream target in potato development.

Among many factors that regulate potato tuberization, calcium and calcium-dependent protein kinases (CDPKs) play an important role. CDPK activity increases at the onset of tuber formation with StCDPK1 expression being strongly induced in swollen stolons. However, not much is known about the transcriptional and posttranscriptional regulation of StCDPK1 or its downstream targets in potato development. To elucidate further, we analyzed its expression in different tissues and stages of the life cycle. Histochemical analysis of StCDPK1::GUS (ß-glucuronidase) plants demonstrated that StCDPK1 is strongly associated with the vascular system in stems, roots, during stolon to tuber transition, and in tuber sprouts. In agreement with the observed GUS profile, we found specific cis-acting elements in StCDPK1 promoter. In silico analysis predicted miR390 to be a putative posttranscriptional regulator of StCDPK1. Quantitative real time-polymerase chain reaction (qRT-PCR) analysis showed ubiquitous expression of StCDPK1 in different tissues which correlated well with Western blot data except in leaves. On the contrary, miR390 expression exhibited an inverse pattern in leaves and tuber eyes suggesting a possible regulation of StCDPK1 by miR390. This was further confirmed by Agrobacterium co-infiltration assays. In addition, in vitro assays showed that recombinant StCDPK1-6xHis was able to phosphorylate the hydrophilic loop of the auxin efflux carrier StPIN4. Altogether, these results indicate that StCDPK1 expression is varied in a tissue-specific manner having significant expression in vasculature and in tuber eyes; is regulated by miR390 at posttranscriptional level and suggest that StPIN4 could be one of its downstream targets revealing the overall role of this kinase in potato development.

The isoflavone fraction from soybean presents mRNA maturation inhibition activity.

Recent findings indicate that mRNA splicing inhibitors can be potential anticancer candidates. We have previously established a screening system which monitors mRNA processing in order to identify mRNA processing inhibitors. Among a number of dietary resources, isoflavone fractions showed an inhibitory effect of mRNA processing. These findings demonstrate that a variety of dietary sources have an impact on mRNA biogenesis.

snRNA 3' End Processing by a CPSF73-Containing Complex Essential for Development in Arabidopsis.

Uridine-rich small nuclear RNAs (snRNAs) are the basal components of the spliceosome and play essential roles in splicing. The biogenesis of the majority of snRNAs involves 3' end endonucleolytic cleavage of the nascent transcript from the elongating DNA-dependent RNA ploymerase II. However, the protein factors responsible for this process remain elusive in plants. Here, we show that DEFECTIVE in snRNA PROCESSING 1 (DSP1) is an essential protein for snRNA 3' end maturation in Arabidopsis. A hypomorphic dsp1-1 mutation causes pleiotropic developmental defects, impairs the 3' end processing of snRNAs, increases the levels of snRNA primary transcripts (pre-snRNAs), and alters the occupancy of Pol II at snRNA loci. In addition, DSP1 binds snRNA loci and interacts with Pol-II in a DNA/RNA-dependent manner. We further show that DSP1 forms a conserved complex, which contains at least four additional proteins, to catalyze snRNA 3' end maturation in Arabidopsis. The catalytic component of this complex is likely the cleavage and polyadenylation specificity factor 73 kDa-I (CSPF73-I), which is the nuclease cleaving the pre-mRNA 3' end. However, the DSP1 complex does not affect pre-mRNA 3' end cleavage, suggesting that plants may use different CPSF73-I-containing complexes to process snRNAs and pre-mRNAs. This study identifies a complex responsible for the snRNA 3' end maturation in plants and uncovers a previously unknown function of CPSF73 in snRNA maturation.

Formate: The Neglected Member of One-Carbon Metabolism.

Formate, the only non-tetrahydrofolate (THF)-linked intermediate in one-carbon metabolism, is produced in mammals from a variety of metabolic sources. It occurs in serum of adults at a concentration of approximately 30 µM. Its principal function lies as a source of one-carbon groups for the synthesis of 10-formyl-THF and other one-carbon intermediates; these are primarily used for purine synthesis, thymidylate synthesis, and the provision of methyl groups for synthetic, regulatory, and epigenetic methylation reactions. Although formate is largely produced in mitochondria, these functions mostly occur in the cytoplasm and nucleus. Formate plays a significant role in embryonic development, as evidenced by the effectiveness of formate in the pregnant dam's drinking water on the incidence of neural tube defects in some genetic models. High formate concentrations in fetal lambs may indicate a role in fetal development and suggest that extracellular formate may play a role in the interorgan distribution of one-carbon groups.

MicroRNA-125b-5p mimic inhibits acute liver failure.

The lack of broad-spectrum anti-acute liver failure (ALF) therapeutic agents contributes to ALF-related mortality. MicroRNAs (miRNAs) are suggested to be potent serum biomarkers for ALF, but their functional and therapeutic relevance in ALF are unclear. Here we show an unbiased approach, using two complementary miRNA screens, to identify miRNAs that can attenuate ALF. We identify miR-125b-5p as a regulator of cell death that attenuates paracetamol-induced and FAS-induced toxicity in mouse and human hepatocytes. Importantly, administration of miR-125b-5p mimic in mouse liver prevents injury and improves survival in models of ALF. Functional studies show that miR-125b-5p ameliorates ALF by directly regulating kelch-like ECH-associated protein 1, in turn elevating expression of nuclear factor-E2-related factor 2, a known regulator in ALF. Collectively, our findings establish miR-125b-5p as an important regulator of paracetamol-induced and FAS-induced cell death. Thus, miR-125b-5p mimic may serve as a broad-spectrum therapeutic attenuator of cell death during ALF.

A New Mutation, hap1-2, Reveals a C Terminal Domain Function in AtMago Protein and Its Biological Effects in Male Gametophyte Development in Arabidopsis thaliana.

The exon-exon junction complex (EJC) is a conserved eukaryotic multiprotein complex that examines the quality of and determines the availability of messenger RNAs (mRNAs) posttranscriptionally. Four proteins, MAGO, Y14, eIF4AIII and BTZ, function as core components of the EJC. The mechanisms of their interactions and the biological indications of these interactions are still poorly understood in plants. A new mutation, hap1-2. leads to premature pollen death and a reduced seed production in Arabidopsis. This mutation introduces a viable truncated transcript AtMagoΔC. This truncation abolishes the interaction between AtMago and AtY14 in vitro, but not the interaction between AtMago and AteIF4AIII. In addition to a strong nuclear presence of AtMago, both AtMago and AtMagoΔC exhibit processing-body (P-body) localization. This indicates that AtMagoΔC may replace AtMago in the EJC when aberrant transcripts are to be degraded. When introducing an NMD mutation, upf3-1, into the existing HAP1/hap1-2 mutant, plants showed a severely reduced fertility. However, the change of splicing pattern of a subset of SR protein transcripts is mostly correlated with the sr45-1 and upf3-1 mutations, not the hap1-2 mutation. These results imply that the C terminal domain (CTD) of AtMago is required for the AtMago-AtY14 heterodimerization during EJC assembly, UPF3-mediated NMD pathway and the AtMago-AtY14 heterodimerization work synergistically to regulate male gametophyte development in plants.